Efficient repair of spontaneous and induced DNA damage is critical for maintaining the mitotic stability of eukaryotic genomes. The proposed experiments will use budding yeast as a model to (1) examine the repair of defined double-strand breaks, (2) examine how transcription leads to instability of the underlying DNA template, and (3) define the nature and mechanism of genetic changes that occur in non-dividing cells. Double-strand breaks (DSBs) are an especially toxic DNA lesion and are repaired by two distinct pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The NHEJ pathway ligates ends that often require processing, which results in loss/gain of sequence at the joint and renders the process highly error-prone. By contrast, HR restores a broken molecule by copying information from an intact chromosome, and thus is considered a high-fidelity process. Even so, HR can alter the linkage of sequences that flank an initiating DSB to result in loss of heterozygosity, or can engage dispersed repeated sequences to generate chromosome rearrangements. Though not required for survival in yeast, both HR and NHEJ are essential in mammals and defects have been linked to a large number of human diseases that include neurological disorders, immune system dysfunction, premature aging syndromes and cancer. Proposed experiments will define molecular intermediates and genetic mechanisms of DSB repair, with a focus on how end structure/sequence affects genetic outcomes. In addition, strand-exchange intermediates associated with spontaneous versus DSB-induced recombination will be examined to resolve a long-standing issue in the field: the relative contribution of DSBs versus single-strand gaps to spontaneous HR. We have shown that transcription destabilizes the underlying DNA template, resulting in locally elevated mutagenesis that alters the mutation landscape across the genome. Transcription-associated mutagenesis (TAM) derives largely from the activity of Topoisomerase 1 (Top1), an enzyme recruited to remove DNA torsional stress created by transcription. Proposed experiments will focus on Top1-dependent mutagenesis and on whether there is an asymmetry between the transcribed and non-transcribed DNA strands in terms of damage and mutation accumulation. Though studies of DSB repair and TAM are traditionally done using exponentially dividing cells, genetic changes also arise in non-dividing cells. Such changes are particularly relevant to alterations that occur in post-mitotic human tissue, which can drive disease and tumor formation. We will examine mutagenesis due to TAM and NHEJ, each of which can be a replication-independent process, in non-dividing yeast cells. Together, these studies will establish new paradigms for the regulation of genome stability in dividing as well as non-dividing eukaryotic cells.